Studies on the oxidative fraction of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. Influence of ligands on its obtention enzymic properties

In the work presented here, we have examined the most favorable conditions that lead to the isolation of the oxidative fraction of glyceraldehyde‐3‐phosphate dehydrogenase by chymotryptic hydrolysis. NAD + as well as glyceraldehyde‐3‐phosphate protect the apoenzyme against proteolysis and therefore do not favor the formation of the oxidative fraction. Nicotinamide mononucleotide, and d ‐glyceraldehyde modify none of the properties related to the kinetics of proteolysis and do not change the yield in oxidative fraction. Because it oxidizes the sulfhydril groups of the active center of the enzyme, o ‐iodosobenzoic acid destroys the protective action of NAD + with regards to proteolysis, but it also decreases the yield in oxidative fraction. Only AMP has been found to render the apoenzyme more sensitive to chymotryptic digestion and to greatly increase the yield in the oxidative fraction. The effects of glyceraldehyde‐3‐phosphate, NAD + and AMP on the modification of the arsenolytic activity during chymotryptic hydrolysis have enabled us to determine the dissociation constants of the enzyme for these three effectors. We have also examined the enzymatic properties of the purified oxidative fraction. Based on the two enzymatic activities retained in the purified oxidative fraction, the following values have been obtained: K m for d ‐glyceraldehyde = 61 μM, K m for NAD + = 120 μM, K m for p ‐nitrophenylacetate = 6.3 μM. The phosphorylating, phosphatase and NADH‐X activities are absent. As a result of proteolysis, it is possible to dissociate three classes of catalytic functions: (1) the phosphorylating, phosphatase and NADH‐X activities, (2) the oxidative activity and (3) the esterase activity.